A major problem in coronary bypass treatment for arteriosclerosis is the lack of suitable autogenous replacement vessels in many patients. This proposal explores new technologies to create tissue-engineered blood vessels (TEBVs) that can serve as readily available replacements for coronary arteries. The bulk of the thickness of an artery wall (the media) is comprised of vascular smooth muscle cells (VSMCs) arranged in concentric layers that provide mechanical strength and contractility. In Aim 1, we will construct TEBV media that incorporate a novel, biocompatible, collagen membrane (CM) scaffold that we developed recently. The CMs' high intrinsic strength and capacity to prealign VSMCs in vitro (via microgrooving) could dramatically shorten TEBV fabrication times to a matter of days, rather than the weeks or months required by other methods. CMs populated with confluent, aligned human coronary artery SMCs (HCASMCs) will be wrapped around a mandrel to form a multilamellar tube. Following removal of the mandrel, the TEBV lumen will be seeded with human coronary artery endothelial cells and the TEBV subsequently grown in a bath of nutrient fluid with nutrient fluid also circulated through the TEBV lumen. After specific intervals of culture, the TEBVs will be evaluated for: (i) deposition of extracellular matrix (ECM) by the VSMCs, (ii) structural changes in the CM scaffolds, (iii) survival, orientation and state of differentiation of the VSMCs, (iv) functional endothelialization of the lumen, and (v) mechanical (burst) strength. The resilient protein elastin is abundant in normal artery walls and its absence in most TEBVs results in a lack of elasticity and excessive proliferation of VSMCs which narrows the TEBV lumen. Ultimately, it would be desirable to use the patient's own VSMCs to limit graft rejection. Unfortunately, adult VSMCs synthesize little or no elastin. To address this problem, Aim 2 evaluates ECM deposition, cell survival and orientation, and mechanical properties (burst-strength, stress/strain response) of TEBVs that incorporate adult rat aortic SMCs that produce elevated levels of elastin as a consequence of expression (via viral transduction) of the proteoglycan versican 3. This proposal seeks to develop new approaches and technologies and apply them to an important problem in human health (coronary artery disease). Moreover, data from this work will serve as a foundation for further development of TEBVs. Accordingly, this application is well suited to the purpose of the R21 mechanism. [unreadable] [unreadable] [unreadable]